PROJECT SUMMARY The enhanced colorectal mechanosensation in colonic hypersensitivity and gastrointestinal (GI) pain is initiated and mediated by primary afferent neurons in dorsal root ganglia (DRG), perhaps by Piezo2-mediated mechanotransduction in a subpopulation of colonic nociceptive afferent neurons. Macrophage colony- stimulating factor (CSF1) and brain-derived neurotrophic factor (BDNF) are two potent mediators in the genesis of mechanical pain, however, DRG nociceptive neurons do not express CSF1 receptor Csf1r and few neurons have BDNF receptor TrkB. Macrophages and glial cells including satellite glial cells (SGCs) express Csf1r and/or TrkB isoforms, therefore they are likely primary recipients of CSF1 and BDNF. In DRG, macrophages and glial cells surround sensory neurons to form a neuronal-glial-immune cell triad. Upon activation, macrophages and glial cells release a variety of inflammatory factors such as the pronociceptive cytokine tumor necrosis factor alpha (TNF?) that can act on nearby sensory neurons. In our preliminary study, macrophages and glial cells act differentially in producing TNF? in DRG of male and female mice in colonic hypersensitivity models. TNF? and CSF1-conditioned human macrophage culture medium activate a subset of Piezo2 expressing colonic afferent neurons. Macrophages are also activated by BDNF to produce TNF?. We postulate that macrophages and glial cells are activated by CSF1 and BDNF in DRG to generate TNF? that activates Piezo2 expressing colonic afferent neurons, leading to colonic mechanosensory sensitization. We will pursue three interrelated Specific Aims. In AIM 1, we will distinguish the roles of macrophages and glial cells in producing TNF? to generate neuroinflammation in DRG in male and female mice to regulate colonic hypersensitivity. We will implement genetic tools for conditional ablation/inhibition of macrophages or glial cells, and examine colorectal distension (CRD)-evoked mechanosensory activity by in vivo DRG imaging. In AIM 2, we will determine Piezo2 in DRG nociceptive neurons in mediation of neuroinflammatory regulation of colonic hypersensitivity. We will use our Piezo2 conditional knockout mouse line to perform direct neurochemical measurement and study the functional roles of Piezo2 in mediation of neuroinflammation (TNF?)-regulated colonic mechanosensory activity. In AIM 3, we will characterize the regulation of macrophages and glial cells by CSF1 and BDNF in male and female mice in producing TNF? to regulate Piezo2 expressing colonic afferent neurons. In many species and systems, the expression of TrkB receptors is sex-related. We will examine whether TrkB receptors are sexually dimorphic in macrophages and glial cells to interpret the distinct roles of macrophages and glial cells in colonic hypersensitivity in male and female mice, and suggest mechanism of gender-related GI pain in patients. Our studies using in vivo, in vitro, genetic tools, and imaging techniques in characterizing the impact of neuroinflammation on colonic mechanosensory activity will open new avenues in designing specific target(s) in the treatment of GI pain and functional bowel disorders.